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Transgenic Papaya
Introduction Papaya is a major cash crop in many tropical and subtropical economies. It is a very nutritious fruit high in Vitamins A, B, and C, as well as beta carotene. The fruit is beneficial to these poorer countries both fiscally and nutritionally. This adds to the need to fight against the massive destruction of papaya by Ringspot virus. The virus is spread by aphids after they have bit into an infected plant. Ringspot virus has a phenotype leading to a prominent mosaic pattern on the leaves, deformed young leaves, watersoaked regions and oily streaks (figure 1). This phenotype allows for detection but PCR and ELISA can be used to confirm. Ringspot Virus Genome The Ringspot Virus has a single stranded RNA genome, about 10,300 nucleotides in length, which encodes a single polypeptide. The single protein is 3,344 amino acids which is cleaved by virus encoded proteases. There are 8 to 9 cleavage sites, yielding 8 proteins, and one protein that may consist of different lengths(figure 2). The eight cleaved proteins, in genomic order, are: P1, HC-Pro, P3, C1, 6k, NIa, NIb, and CP. P1 self-cleaves and is involved in cell-to-cell movement of the virus. Helper component (HC-Pro) is also a protease that is necessary for viral infection. It is involved in cell-to-cell movement, long distance movement, symptom expression, transmission, genome amplification, and suppression of RNA silencing. The role of P3 is unknown but may be involved in replication. Cylindrical inclusion protein (C1) is an RNA helicase involved in genome replication. It has an NTPase active site as well. The function of 6K is also unresolved but may be involved in replication as a well as inhibiting nuclear inclusion protein (NIa) transport to the nucleus. the NIa protein has two domains a VPg and a Pro domain. NIa-VPg is the primer for RNA synthesis initiation. NIa-pro is the third encoded protease. NIb is an RNA polymerase. NIa and b are essential for infection. The last protein, coat protein (CP), is not essential for infection. It is involved in RNA encapsulation, transmission, and systemic movement. These proteins make up 94.5% of the virion mass and the remaining mass is from nucleic acid. Transgenic Disease Management Traditional methods to try to control the spread of Ringspot Virus were tried and abandoned. The first method tried was selective breeding. There are wild strains of papaya that are resistant, but unfortunately they are unable to breed and produced offspring. Cross protection was also attempted, this is similar to getting a live vaccine. In a cross protection study, the plant is exposed to a mild form of the virus. Due to the large variability between Ringspot strains, cross protection proved to be difficult. Transgenetic resistance can have two approaches, protein mediated or RNA mediated. Both methods have been utilized with three different targets. The first target was the coat protein, mimicking transgenic tobacco. The Ringspot virus resistant coat protein is transferred into a zygotic papaya embryo via a plasmid with neomycin phosphotransferase II gene. Variations of this method have been employed because the effectiveness is dependent upon the strain (Table 1). To try to find a more generic approach, RNA Interference (RNAi) mediated resistance was attempted, again based upon tobacco plants. RNAi is used to inhibit the expression of certain genes. In this method the transgene need to be very similar to the target gene. Researchers targeted the HC-Pro gene for silencing. HC-Pro is responsible for posttranscriptional gene silencing, the over expression of 21-25 siRNAs that target papaya mRNA. The inhibition of HC-pro has lead to Ringspot Virus resistance in transgenic papaya. Again, researchers adapted methods used in tobacco plants to attempt protein mediated resistance. In this method they inserted replicase genes with mutations. This method led to high resistance. References 1. Azad, AK, Amin L, and Sidik NM. Gene Technology for Papaya Ringspot Virus Disease Management. The Scientific World Journal. 2014. PMID 24757435 2.Gonsalves, D., S. Tripathi, J. B. Carr, and J. Y. Suzuki. 2010. Papaya Ringspot virus. The Plant Health Instructor. DOI: 10.1094/PHI-I-2010-1004-01 http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/PapayaRingspotvirus.aspx